The present application is the second competing renewal of a CCHI grant first awarded in 2003 to study human immune response to Bacillus anthracis and the toxin vaccine that is currently administered to the thousands of deployed US military. The goals of the original application were: (a) To study the human immune response to a challenging vaccine, (b) To understand the mechanism for the high lethality of the inhalation form of the disease, (c) To understand the cellular basis of the host response to the pathogen. Our published findings show, in contrast to prevailing views, that the high rate of mortality in inhalation anthrax is due to bacterial sepsis and not the anthrax toxins. In the most recent funding cycle, we made the seminal discovery that immune complexes between peptidoglycan (PGN) and pre-existing anti-PGN antibodies present in all humans may be the source of the massive inflammation accompanying infection by B. anthracis. Lastly, we discovered that fully 30% of vaccinees are almost certainly unprotected despite having been immunized more than 6 times in an onerous vaccine schedule. In this renewal application, we will follow up on these exciting discoveries to determine: (a) How do anthrax spores move from the alveoli to the mediastinal lymph nodes. (b) How PGN triggers an antimicrobial response by human immune cells and how anthrax toxins affect the response. (c) How PGN-anti-PGN immune complexes influence the outcome of the disease. (d) Why the vaccine is imperfect in stimulating the maturation of germinal center B cells, and whether elevated histones caused by toxin-impaired efferocytosis contributes to inflammation. Our work is supported by 3 scientific cores: An animal core that applies a non-human primate model established in the previous funding cycle; a flow cytometry core that has state-of-the-art capacity; and a human antibody core that will make recombinant human monoclonal antibodies using technology that was invented at our institute. The studies in this renewal application-are focused and thematically organized around the key roles of PGN, the anthrax toxins and the anthrax toxin vaccine in the human immune responses. They have great potential to identify novel means of interrupting the pathology caused by this model Gram-positive pathogen. RELEVANCE: B. anthracis is highly relevant and important agent of bioterrorism. Although a vaccine is provided to military personnel, it is not given to private citizens and is of highly questionable efficacy. We have made great progress in understanding B. anthracis infections in humans and propose novel studies to expand our published findings. Project 1: Toxin-Mediated Suppression of Human PBMC Responses During Bacteremia Project Leader (PL): Ballard, Jimmy DESCRIPTION (provided by applicant): Bacteremia can lead to septic shock and death in infected individuals, yet many aspects of bacteremia remain poorly understood in humans. In particular the host responses to bacterial components and the suppression of these responses by virulence factors in humans mononuclear cells has not been fully investigated and this has limited the development of new therapeutics. Using Bacillus anthracis as a model system we are investigating inflammatory stimuli (peptidoglycan) and modulation of responses to this stimuli by anthrax toxin. In particular we are characterizing the induction of antimicrobials by PGN and studying how anthrax lethal toxin, but not edema toxin, suppresses this response. The first aim of this project is to characterize the BaPGN-induced antimicrobial response and its modulation by anthrax toxin in human PBMCs. The second aim is to characterize the differential mechanisms by which LT and ET modulate inflammatory and antimicrobial responses. The final aim is to examine the interplay between BaPGN-induced proinflammatory/antimicrobial factors and their suppression by anthrax toxin in an in vivo model. Completing these aims will address our overall goal of defining the molecular and cellular events occurring between a gram positive pathogen that causes bacteremia and the host responses to that pathogen. RELEVANCE: Bacteremia and septic shock are serious medical conditions and treatment options are limited. Results from our studies will provide information about this disease event, with a specific focus on this process in humans and possible ways to treat infected individuals.